Satiation peptide administration

ABSTRACT

Disclosed herein are compositions and methods for treating obesity involving satiation gut peptide administration to the mouth of a subject for a predetermined dose and frequency. In other embodiments, materials and methods of treating certain psychological disorders are disclosed involving satiation gut peptides. In exemplary embodiments, the satiation gut peptide pertains to PYY.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 61/146,287 filed Jan.21, 2009, which is incorporated herein in its entirety.

INTRODUCTION

Satiation gut peptides are secreted from the small intestine and colonin response to food intake. Penetrating from plasma through theblood-brain barrier, they act by activating specific receptors in thesatiety center of the hypothalamus thus inducing satiation. The mostimportant satiation gut peptides are Peptide YY, Glucagon-like Peptide1, Oxyntomodulin, and Cholecystokinin. Acute supplemental therapy withsatiation gut peptides reduces food intake and body weight in obeseanimal models as well as in lean and obese human subjects. Severalclinical trials utilizing satiation peptide supplement therapy arecurrently under way. In these trials, the tested peptide is administeredby iv injections 30 min prior to the meal. It is widely acknowledgedthat satiation gut peptides would not be effective through ingested oraladministration since enzymes and acids in the gut would degrade themprior to reaching the blood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: rAAV-PYY vector plasmid: TR (terminal repeat), CMV(cytomegalovirus) enhancer, CBA (Chicken B actin) promoter.

FIG. 2:. Food intake per week in 8 months-old DIO mice injected withrAAV-PYY vs. rAAV-GFP*P<0.05

FIG. 3.: Body weight change in 8 months-old diet-induced obese(DIO)-mice injected with rAAV-PYY vs. rAAV-GFP (controls). *P<0.05

FIG. 4: Effects of Peptide YY Mouth Spray. A) One hour food intake afterone spray with PYY, 5 ug per 100 g of body weight vs. spray with sterileH₂O. B) Difference in one hour food intake after PYY mouth spray vs.Sterile H2O, shown are individual animals. C) Dose response of PYY mouthspray on FI as measured after one hour. D) 24 hours food intake afterPYY mouth spray vs. sterile H₂O. *P<0.05, **P<0.01.

FIG. 5: relates to effects of extended PYY administration on behavior ofmice concerning (A) attack frequency (B) threat frequency (C) chasefrequency and (D) frequency.

FIG. 6: shows example sequences pertaining to a) Human Peptide YY aminoacid sequence, b) a portion of human PYY, c) Oxyntomodulin polypeptide,d) Glucagon-like Peptide 1, and e) cholecystokinin related sequences.

DETAILED DESCRIPTION

The present application is based on the inventors' work towardestablishing either a stable and longer term delivery of satiation gutpeptides and/or administration of satiation gut peptides intended fortargeting specific areas of the body which have now been identified aseffecting an unexpectedly favorable satiation response. According tocertain embodiments, the invention pertains to compositions and methodsfor treating obesity involving satiation gut peptide administration tothe mouth of a subject for a predetermined dose and frequency. Accordingto specific embodiment, the subject invention pertains to providing along-term increase of satiation peptides in the saliva by targetingsalivary gland with vectors, including, but not limited to, recombinantAdeno-associated viral (rAAV) vector, adenoviral vector, or othersuitable vectors for transfection of cells in a human or non-humananimal, harboring the respective gene, for introduction and expressionin targeted cells.

In another embodiment, the invention pertains to a method of inducingsatiation in a subject that includes applying to at least a portion ofthe mouth of the subject a composition comprising a satiation gutpeptide at a time period prior to eating (pre-prandial). The time periodmay be 5 seconds or more. In a specific embodiment, the time period is5-360 min prior to eating. In a more specific embodiment, the timeperiod is 30-120 min prior to eating.

Another embodiment relates to a container that comprises a solid (e.g.powder), fluid or semi-fluid composition that comprises satiation gutpeptide and a pharmaceutically acceptable carrier. In a specificembodiment the container comprises a nozzle for ejecting the compositioninto the mouth of a subject. The container may be under pressure and/orbe equipped with a pump nozzle.

Another embodiment relates to a mouth applicable article loaded with asatiation gut peptide. The article may be chewing gum loaded withpeptide; a lozenge (e.g. a dissolvable solid or semi-solid objectintended to hold in the mouth for a period of time) loaded with peptide,or a permeable pouch or sponge loaded with peptide. The article isdesigned for extended delivery of peptide to the mouth and/or pharynx,as opposed to conventional oral administration that involves theimmediate swallowing of a pill, table or fluid composition as isconventionally understood as oral adminstration. In particular, thearticle is designed for delivery to the tongue.

According to another embodiment, cells related to the mouth such asmucosal or salivary gland cells are transformed with vectors engineeredto express and release a satiation gut peptide.

In a specific embodiment, the peptide is delivered to the mouth and/orpharynx to a subject according to a generally continuous time period ofat least 5, 10, 15 or more seconds. In another embodiment, the deliveryis for 0.1-120 mins, including any specific 0.1 minute increment withinsuch range. In a specific embodiment, the inventors have found thatadministration of the peptide such that it is in prolonged contact withthe tongue is optimal.

As described herein, the invention includes embodiments that utilizenucleotides encoding satiation gut peptides, or peptides alone.Satiation gut peptides include peptides relating to Peptide YY,Glucagon-like Peptide 1, Oxyntomodulin, and cholecystokinin. Nucleotidesand peptides having substantial identity to the nucleotide and aminoacid sequences relating to Peptide YY, Glucagon-like Peptide 1,Oxyntomodulin, and cholecystokinin also are contemplated for use inaccordance with the teachings herein. Sequence information is providedin FIG. 6.

The proteins and polypeptide sequences, as well as polynucleotidesencoding the same, having substantial identity with the sequencesspecifically described herein may be used in conjunction with presentinvention. Here “substantial identity” means that two sequences, whenoptimally aligned such as by the programs GAP or BESTFIT (peptides)using default gap weights, or as measured by computer algorithms BLASTXor BLASTP, share at least 50%, preferably at least 75%, and mostpreferably at least 95% sequence identity, or sequence identity of anyinteger percentage between 50% and 99.9%. Preferably, residue positionswhich are not identical differ by conservative amino acid substitutions.For example, the substitution of amino acids having similar chemicalproperties such as charge or polarity are not likely to effect theproperties of a protein. Non-limiting examples include glutamine forasparagine or glutamic acid for aspartic acid.

The term “variant” as used herein refers to nucleotide and polypeptidesequences wherein the nucleotide or amino acid sequence exhibitssubstantial identity with the nucleotide or amino acid sequence SEQ IDNOS, preferably 75% sequence identity and most preferably 90-95%sequence identity to the sequences of the present invention: providedsaid variant has a biological activity as defined herein. The variantmay be arrived at by modification of the native nucleotide or amino acidsequence by such modifications as insertion, substitution or deletion ofone or more nucleotides or amino acids or it may be a naturallyoccurring variant. The term “variant” also includes homologous sequenceswhich hybridise to the sequences of the invention under standard orpreferably stringent hybridisation conditions familiar to those skilledin the art. Examples of the in situ hybridisation procedure typicallyused are described in (Tisdall et al., 1999); (Juengel et al., 2000).Where such a variant is desired, the nucleotide sequence of the nativeDNA is altered appropriately. This alteration can be made throughelective synthesis of the DNA or by modification of the native DNA by,for example, site-specific or cassette mutagenesis. Preferably, whereportions of cDNA or genomic DNA require sequence modifications,site-specific primer directed mutagenesis is employed, using techniquesstandard in the art.

In specific embodiments, a variant of a polypeptide is one having atleast about 80% amino acid sequence identity with the amino acidsequence of a native sequence full length sequence of satiation gutpeptides as taught herein and known in the art. Such variantpolypeptides include, for instance, polypeptides wherein one or moreamino acid residues are added, or deleted, at the N- and/or C-terminus,as well as within one or more internal domains, of the full-length aminoacid sequence. Fragments of the peptides are also contemplated.Ordinarily, a variant polypeptide will have at least about 80% aminoacid sequence identity, more preferably at least about 81% amino acidsequence identity, more preferably at least about 82% amino acidsequence identity, more preferably at least about 83% amino acidsequence identity, more preferably at least about 84% amino acidsequence identity, more preferably at least about 85% amino acidsequence identity, more preferably at least about 86% amino acidsequence identity, more preferably at least about 87% amino acidsequence identity, more preferably at least about 88% amino acidsequence identity, more preferably at least about 89% amino acidsequence identity, more preferably at least about 90% amino acidsequence identity, more preferably at least about 91% amino acidsequence identity, more preferably at least about 92% amino acidsequence identity, more preferably at least about 93% amino acidsequence identity, more preferably at least about 94% amino acidsequence identity, more preferably at least about 95% amino acidsequence identity, more preferably at least about 96% amino acidsequence identity, more preferably at least about 97% amino acidsequence identity, more preferably at least about 98% amino acidsequence identity and yet more preferably at least about 99% amino acidsequence identity with a polypeptide encoded by a nucleic acid moleculeshown in Attachment B or a specified fragment thereof. Ordinarily,variant polypeptides are at least about 10 amino acids in length, oftenat least about 20 amino acids in length, more often at least about 30amino acids in length, more often at least about 40 amino acids inlength, more often at least about 50 amino acids in length, more oftenat least about 60 amino acids in length, more often at least about 70amino acids in length, more often at least about 80 amino acids inlength, more often at least about 90 amino acids in length, more oftenat least about 100 amino acids in length, or more.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA tore-anneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired identitybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by those that: (1) employ low ionic strength andhigh temperature for washing, 0.015 M sodium chloride/0.0015 M sodiumcitrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, 50% (v/v) formamide with 0.1% bovineserum albumin/0.1% Ficol/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodiumcitrate at 42 degrees C.; or (3) employ 50% formamide, 5×SSC (0.75 MNaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5.times. Denhardt's solution, sonicated salmonsperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42 degreesC., with washes at 42 degrees C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55 degrees C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55 degreesC.

“Moderately stringent conditions” are identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37.degree. C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50 degrees C. Theskilled artisan will recognize how to adjust the temperature, ionicstrength, etc. as necessary to accommodate factors such as probe lengthand the like.

An oral liquid formulation may, for example, be a pharmaceuticallyacceptable emulsion, syrup, elixir, suspension, solution and the like,which may contain a pharmaceutically customary inert diluent such aswater and if desired, additives. Such an oral liquid formulation can beproduced by mixing an active ingredient, inert diluent and otheradditives if necessary in accordance with a customary method. An oralformulation usually contain about 0.01 to 99% by weight, preferablyabout 0.1 to 90% by weight, usually about 0.5 to 50% by weight of aninventive active compound, although the amount may vary depending on thedosage form.

In certain embodiments, a formulation is prepared for spraying into themouth. The composition may be placed in a container equipped with asprayer nozzle and either ejected through a pump motion or by release ofpressure.

In another embodiment, the composition is combined and provided in theform of a chewing gum.

Example 1 PYY Gene Therapy

Peptide YY (PYY) is a satiation gut peptide secreted from theneuro-endocrine L cells from the small intestine and colon epithelia.PYY is secreted into the blood stream and subsequently activates Yreceptors in the arcuate nucleus of the hypothalamus thus inducingsatiation. In the pilot study, in addition to the plasma, we have alsodetected PYY in saliva in naïve, untreated mice, while at the same timeidentifying the respective Y2 receptor in the tongue epithelia. To theinventors' knowledge, these are novel findings which lead to thefollowing hypothesis: long-term expression of genes coding for satiationpeptide in salivary gland will reduce food intake and body weight inobese animal.

In order to overexpress PYY, a rAAV vector was constructed harboring thepre-pro-Peptide YY gene (FIG. 1). rAAV-PYY was administered into thesalivary glands through the salivary ducts. Injection of rAAV-PYYresulted in a long-term (up to 6 months) expression of Peptide YY asmeasured by the respective ELISA assay (not shown).

The ectopic expression of PYY transgene in lean as well as indiet-induced obese mice produced a significant decrease in food intakeand body weight compared to control group injected with reporter vectorrAAV-GFP (FIGS. 2 and 3). The results of the inventors demonstrated thatlong-term expression of peptide YY transgene delivered by a viral vectoris a viable therapy for the treatment of obesity.

Example 2 Long-term Peptide YY Gene Therapy: Addressing ExistingControversy

Peptide YY (PYY) is a satiation gut hormone released postprandiallymainly by the gut. The effects of acute and chronic administration ofPYY are controversial. Several groups have found a significant decreasein food intake (FI) and body weight (BW) in animal experiments and inhuman trials, while other groups have been unable to reproduce thisdata. The controversy can be related to several behavioral factorsincluding acclimatization and stress, as well as varying experimentalconditions. To eliminate these factors and to address the effect oflong-term overexpression of PYY we have develop animal models, C57BL/6mice, with either homotopic or ectopic expression of pre-pro-PYYtransgene delivered by a single injection of a viral vector. For theenhanced homotopic expression, the vector was delivered through superiormesenteric artery (SMA) to target the colon and small intestine wherePYY is normally produced. For the ectopic expression, the vector hadbeen delivered either into the 3^(rd) ventricle in the brain targetinghypothalamus, or into the salivary ducts to target submandibularsalivary glands to induce PYY secretion in to saliva. All treated micewere fed high fat diet (60% fat) ad libitum, FI and BW were measuredonce a week for 30 weeks. In SMA-injected mice, we documented asustained two-fold increase of PYY in plasma during fasting and ten-foldincrease one hour after feeding. In spite of the significant increase ofsystemic PYY, no differences in BW or FI were documented at 30 weekspost-injection. On the contrary, in mice with PYY-encoding vectorinjected either centrally or in the salivary glands, the concentrationof plasma PYY remained unchanged. However, centrally-injected miceexhibited significant increase in both BW and FI, while the long-termeffect was opposite in salivary gland-treated animals. In satiationbehavioral studies, neither treated group show a significant differencein FI after 16, or 24 hrs fasting. Our results suggest that thelong-term overexpression of PYY have differential effect dependent onthe targeted site. Physiological tests and hormonal profiles in micefrom all groups will be presented and the possible mechanism of actionof the exogenous PYY will be discussed.

Example 3 Administration of Satiation Gut Peptides to Mouth Introduction

Satiation gut peptides are secreted into the bloodstream from the smallintestine and colon in response to food intake (FI). Their main effectis to induce satiety by activation their specific receptors in thesatiety center in the hypothalamus. The most important satiation gutpeptides are Peptide YY (PYY), Glucagon-like Peptide 1 (GLP-1),Oxyntomodulin (OXM), and Cholecystokinin (CCK). Acute supplementaltherapy with satiation gut peptides reduces FI and body weight (BW) inobese animal models as well as in lean and obese human subjects. Severalclinical trials utilizing satiation peptide supplement therapy arecurrently under way. Unfortunately, the delivery methods of thesepeptides (iv injections) showed significant side effects and pooradherence. In the pilot study, in addition to the plasma, we have alsodetected PYY in saliva in naïve, untreated mice, while at the same timeidentifying the respective Y2 receptor in the tongue epithelia. Based onthese novel findings, the inventors have developed a non-invasive,easy-to-use mouth spray to deliver these peptides. The aim is to reducevoluntary FI by inducing an early satiation effect mediated by anincreased concentration of these peptides in the saliva. Incrementalreduction in FI over the prolonged period of time will result in reducedBW and improved health.

Materials and Methods:

Synthetic PYY was purchased from Bachem, Inc USA (Cat # H-6042) anddiluted in sterile H₂O. Sterile un-used perfume sample vials (Saphora)were utilized to administer PYY in the form of a mouth spray. It wasestimated that the volume of one spray approximates to about 25 l.

Mice were conditioned three times to 24 hours fasting starting at thebeginning of the dark cycle and ending at the end of the light cycle. Atthe end of the fasting cycle and as a part of conditioned routine, asterile water spray had been administered into the mouth. All theexperiments were done during the first hour of the dark cycle afterfasting. Once the dark cycle started, mice were sprayed once with eitherPYY or sterile H₂O in a total volume of 25 l per spray. After thetreatment, mice were returned to their cages and ten minutes laterpre-weighted chow was provided. One hour later, the amount of consumedchow had been recorded by measuring the leftover amount. When theexperiments were repeated, mice were fasted only once a week with thecontrol and experimental groups rotated.

Results:

Mice sprayed with PYY consumed significantly (P=0.03) less food (15% onaverage) compared to the control group sprayed with H₂O (FIG. 4A, 4B).We also documented a significant PYY dose response effect to FI (FIG.4C). After PYY mouth spray, there was a pronounced early satiety effectfollowed by compensatory higher food intake resulting in similar overall24 hr period FI for both experimental and control groups (FIG. 4D). Thisdata correlates with previously published observation showing nodifference in FI during 24 hours after IP or IV injections. This data,however, reflect the ad libitum pattern of food consumption in mice. Inhumans, with a defined pattern of three meals per day, with PYY sprayapplication prior to each meal, the treatment is anticipated to reduceoverall FI over 24 hrs period.

Conclusions:

The increase in PYY concentration delivered by mouth spray has apotential to be utilized as a treatment for obesity by reducingvoluntary FI. Attachment A in U.S. Provisional Application No. 61/146287sets forth data demonstrating the successful expression of Peptide YYand the use for modulating BMI and FI.

Example 4 Long-term Salivary PYY3-36 Treatment Modulates AggressiveBehavior

The NPY pathway modulates food intake, body weight, energy expenditure,blood pressure, cortical excitability, circadian rhythms, stressresponse, emotions, memory, attention, learning, aggression, ethanolsusceptibility and pain processing. The NPY pathway has also beenrelated to the mechanism of epilepsy, neurogenesis, neuroprotection,analgesia, anxiety and depression (1, 2). The widespread effects of NPYare mediated by G-protein coupled receptors Y1, Y2, Y4, Y5 and Y6.

Components of the Neuropeptide Y (NPY) expressed widely in the CNS havebeen linked to aggression, anxiety and depression. For example, NPY Y1and Y4 receptor knockout mice exhibit abnormally aggressive behavior(1). Furthermore, both pharmacological inhibition of NPY Y2 receptor andNPY Y2 receptor knockout show an anxiolytic, antidepressant phenotypeswith reduced attention and increased impulsivity (3, 4)'(5). However, sofar little is known about the role of NPY Y2 receptors in aggressivebehavior.

NPY Y2 receptors endogenous agonist is PYY₃₋₃₆. Recently, we reportedthat augmentation of salivary PYY₃₋₃₆ modifies feeding behavior in mice.The long-term increase of salivary PYY₃₋₃₆ by using a recombinantAdeno-associated virus (rAAV-PYY), produced a significant decrease inbody weight and food intake in obese mice. Unexpectedly, in addition tomodulating the feeding behavior, the long term over-expression ofsalivary PYY₃₋₃₆ also appears to modulate aggressive behavior.

Data presented in this report indicate that long-term expression ofPeptide YY₃₋₃₆, an agonist of NPY Y receptors with higher affinity forthe Y2 receptor, abolish aggressive behavior in mice. To test theseobservations, we used the territorial Resident/Intruder (R/I) aggressiveparadigm (6), a standard test for evaluating rodent aggressive behavior.The test was applied on three different occasions using differentintruders. Tests were recorded and analyzed in a blind manner using theObserver v5.0 software (Noldus Information Technology) (see videos,Supplementary data) (7).

The aggressive behavior was analyzed by the frequency, duration andlatency of attacks, threats and chase from the resident to the intrudermice. PYY₃₋₃₆ treated mice displayed a 44-fold decrease in the number ofattack events compare to controls [PYY₃₋₃₆ 0.07±0.067 events per 10 min,vs. Controls 3.07±1.74 events in 10 min, n=5, p<0.05) (FIG. 5A).Likewise, PYY₃₋₃₆ treated mice had a significant decrease in attackduration and a significant increase in attack latency. Similarly,PYY₃₋₃₆ treated mice had a significant decrease in threat events andduration compare to controls (FIG. 5B) and a decrease in chase eventsand duration compare to control mice (FIG. 5C). Interestingly, eventhough an aggressive behavior was almost completely abrogated, thenormal social interactions manifested by sniffing did not change (FIG.5D).

These dramatic changes in territorial aggression suggest that thelong-term treatment with NPY Y2 receptors agonists such as PYY₃₋₃₆modulates both feeding and aggressive behaviors. Because PYY₃₋₃₆ hasrecently been tested in clinical trials for weight loss in obese adultsubjects, the unintended while favorable effects shown here must betaken in consideration before such agonists are approved for thelong-term treatment of obesity. This is especially important in light ofthe Y receptors cross talk and interactions as shown in geneticallymodified mice models (8). Further studies are needed to understand thelong-term effect of Y receptors agonists in feeding and aggressivebehavior, as well as in depression and anxiety.

Supplemental Material: Methods

Vector design: A recombinant adeno-associated virus encoding murinepre-pro-PYY (rAAV-PYY) under the control of a strong constitutiveCMV/-actin promoter and the control rAAV-GFP were pseudotyped into rAAVserotype 5 capsids as having higher transduction in salivary glands (SG)(9). The production, purification and titration of the viral vectorswere performed as described previously (10).

Mouse studies: This study was approved by the Animals Care and UseCommittee of The National Institute of Dental and Craniofacial researchand by the Biosafety Committee of the National Institute of Health(Bethesda, Md.). All mice procedures were done in accordance with theprinciples of the National Research Council's guide for the Care and Useof Laboratory Animals. Studies were done in male Balb/c (Harlan SpragueDawley, Walkersville, Md.) mice housed at 22-24° C. in a 12 hourslight/dark cycle (lights off at 1800). Forty five days old male Balb/Cmice (n=5) were administered a single dose of (100 l, 10¹⁰ vectorgenomes) rAAV-PYY, rAAV-GFP or saline control bi-laterally into theorifice of the submandibular salivary gland as described by Katano et al(9).

Metabolic profile: Mice had free access to water and food (normal chow).Food intake and body weight were measured weekly for 24 weeks.

Behavioral studies: Aggression territorial-Intruder test were performedon week 24 after the treatment (6). Briefly, PYY-, or GFP-treatedresident mice were individually housed for at least two weeks prior totesting. Bedding from cages was not changed during the testing period toavoid unnecessary stress. On the day of the experiment, a smaller sizeintruder was placed into the resident cage for 10 minutes and theresident's behavior was recorded with a video camera. Each experimentwas repeated 3 times on three different occasions and with differentintruders. The videos from the experiments were analyzed fornon-aggressive and aggressive behavior by an expert in a blind mannerusing The Observer v5.0 software (Noldus Information Technology) (7).

Statistical analysis: Statistical analysis was conducted using un-pairedStudent's t-test or by a Mann-Whitney test with significance at P<0.05.Data was reported in mean±SEM.

Results:

Metabolic Profile: rAAV-PYY treated mice weekly caloric intake wassignificantly lower than rAAV-GFP control mice (rAAV-PYY 95.53±2.35 kcalvs. rAAV-GFP 107.44±3.22 kcal, p<0.002). Twenty two weeks after vectordelivery, the rAAV-PYY treated mice gained significantly less weightthan the controls mice (rAAV-PYY 5.33±0.63 g vs. rAAV-GFP 6.28±0.68 g,p<0.05). These data suggest that long-term chronic elevation of PYY₃₋₃₆in saliva of lean mice modulates feeding behavior by decreasing foodintake and body weight.

REFERENCES CITED FOR EXAMPLE 4

1. T. Karl, H. Herzog, Peptides 28, 326 (February 2007).

2. E. E. Benarroch, Neurology 72, 1016 (Mar. 17, 2009, 2009).

3. A. Tschenett et al., Eur J Neurosci 18, 143 (July 2003).

4. J. P. Redrobe, Y. Dumont, H. Herzog, R. Quirion, Behav Brain Res 141,251 (May 15, 2003).

5. B. Greco, M. Carli, Behav Brain Res 169, 325 (May 15, 2006).

6. T. Karl et al., Proc Natl Acad Sci USA 101, 12742 (Aug. 24, 2004).

7. A. M. Muehlmann, B. D. Brown, D. P. Devine, J Pharmacol Exp Ther 324,214 (Jan. 1, 2008, 2008).

8. W. Wittmann, S. Loacker, I. Kapeller, H. Herzog, C. Schwarzer,Neuroscience 136, 241 (2005).

9. H. Katano et al., Gene Ther 13, 594 (April 2006).

10. S. Zolotukhin et al., Methods 28, 158 (October 2002).

The teachings of the references cited throughout the specification areincorporated herein in their entirety by this reference to the extentthey are not inconsistent with the teachings herein. It should beunderstood that the examples and the embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

1. A method of inducing satiation in a subject comprising applying asatiation gut peptide to a mouth of the subject.
 2. The method of claim1, wherein said applying comprises spraying a fluid compositioncomprising a satiation gut peptide into the mouth of the subject suchthat said composition contacts a tongue of the subject.
 3. The method ofclaim 1, wherein applying occurs a period of time prior to eating.
 4. Acontainer comprising a composition that comprises a satiation gutpeptide and a spray nozzle for spraying said composition out of saidcontainer.
 5. A lozenge comprising a composition that comprises asatiation gut peptide.
 6. The lozenge of claim 5, wherein said lozengeis comprised of a dissolving material.
 7. The lozenge of claim 6,wherein said lozenge is in the form of a dissolvable planar sheet, orsolid or semi-solid candy.
 8. A piece of chewing gum comprising asatiation gut peptide.
 9. A vector comprising an expression cassettethat includes a polynucleotide sequence that encodes a satiation gutpeptide.
 10. The vector of claim 9, wherein said expression cassettetransfects salivary gland cells.
 11. A method of increasing satiationgut peptide concentrations in a subject comprising delivering a vectorof claim 9 to a cell of said subject, wherein said satiation gutpolynucleotide sequence is expressed.
 12. A method of treating apsychological disorder of a subject comprising delivering a therapeuticeffect dose of a satiation gut peptide to a mouth of said subjectwherein said satiation gut peptide is available in the mouth for atleast 10 seconds per dose.
 13. The method of claim 12, wherein doses areadministered daily or every other day for at least a week.
 14. Themethod of claim 13, wherein doses are administered daily or every otherday for at least 2 weeks.
 15. The method of claim 13, wherein doses areadministered daily or every other day for at least one month.
 16. Themethod of claim 13, wherein doses are administered daily or every otherday for at least 2 weeks.
 17. The method of claim 12, wherein saidsubject is a human or non-human mammal.
 18. The method of claim 12,wherein said psychological disorder is depression, anxiety, or panicdisorder.
 19. The method of claim 12, wherein said psychologicaldisorder is higher than normal aggression, wherein said method lowersaggressive behavior of said subject.
 20. The method of claim 11, whereinsaid cell is a salivary gland cell, oral epithelial cell or tongue cell.21. A method of treating obesity of a subject comprising delivering atherapeutic effect dose of a satiation gut peptide to a mouth of saidsubject such that said satiation gut peptide is available in the mouthfor at least 30 seconds per dose.
 22. The method of claim 21, whereindoses are administered daily or every other day for at least a week. 23.The method of claim 21, wherein doses are administered daily or everyother day for at least 2 weeks.
 24. The method of claim 21, whereindoses are administered daily or every other day for at least one month.